Antibacterial-based Hand Sanitizer of Biwa Leaves’ Extract

(Eriobotrya japonica (Thunb) Lindl) from Tanah Karo

Irfan Asahan

, Slamet Silaban

, Arif M. Harahap

, Dwi Suryanto

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Department of Pharmacy, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Antibacterial, Biwa (Eriobotrya japonica (Thunb) Lindl.), l, Hand sanitizer.

Abstract: Biwa (Eriobotrya japonica (Thunb) Lindl.) is one of cultivated plant commodities in Karo regency, North

Sumatera, which has been known to contain chemical compounds such as flavonoid and tannin possessing

antibacteria properties. The study aimed to develop the potency of Biwa leaves’ extract as hand sanitizer to

inhibit the growth of Staphylococcus aureus. Methodologies in this study were extraction and detection of

common phytochemical compounds, antibacterial test against S. aureus in vitro, toxicity test using Artemia

salina, characterization of gel formulation, antibacterial test of gel formulation against S.aureus and

antibacteria performance from formulated hand sanitizer. The phytochemical compounds detected in extract

were from flavonoid and tannin groups with antibacterial activity observed at concentration of 15 and 20%

with 8.0 and 9.6 mm diameter of inhibition zones respectively.The Lethal Concentration 50 (LC

) value was

obtained at 162,18 ppm while administration of hand sanitizer reduced S.aureus up to 17.4 colonies in

average.

1 INTRODUCTION

Hands are the frequent mediators to microbes (virus,

fungi and bacteria) in environment during our daily

activities (Wijaya., 2013). Practicing good sanitation

is the best solution to prevent the microbial

contamination to hands. Hand sanitizer is one product

made from ethanol to cleanse the hands, yet

preventing or reducing microbial contamination.

The use of ethanol effectively kills bacteria since

of its bactericidal properties but adverse effects can

be harmful from prolonged use such as burns, dry

skins and irritations (Dewi., 2016). As an alternative,

triclosan may be used as ethanol substitute because of

its less corrosiveness although in some cases, may

also induce bacterial resistance. Therefore, a solution

to address this problem is sought by incorporating

Biwa extract as active ingredient in our formulated

hand sanitizer.

Biwa, also known as loguat, is one of the plant

commodities with potential economical value yet still

rarely cultivated in Indonesia. In North Sumatera, the

plant has been known to be cultivated in Karo

Regency. The Karonese believe that in addition to its

high economical value, Biwa also efficacious as a

drug. Plant parts of Biwa such as fruits have high

antioxidant content, while seeds and leaves were

efficacious as medicine (Morton, 2001). Other local

people in North Sumatra, the Chinese, consumed

Biwa drugs, especially the seeds and leaves for the

treatment of diarrhea, toxin neutralization and

swelling. The fruits were also used as tranquilizer,

skin cosmetic, and cholestero-lowering therapy

(Sembiring, 2009).

Previous study reported the phytochemicals

detected in Biwa leaves containing compounds such

as oleanolic, ursolic acid and megastigmane

glycosides with biological properties as antiviral,

antitumor, hypoglycemic, anti-inflammatory, and

antibacteria (Singh., 2010). Biwa leaves are effective

against bacterial and viral infections in internal bodies

such as bronchitis and gastrointestinal disease

(Brown, 1999). Testing of Biwa leaves externally

asactive ingredient in formulated hand sanitizer is not

much done yet. Based on its potential, the study will

then evaluate the antibacterial properties from Biwa

leaves’ extract against Staphylococcus aureus as well

as obtaining the LC

value from its active fraction to

1076

Asahan, I., Silaban, S., Harahap, A. and Suryanto, D.

Antibacterial-based Hand Sanitizer of Biwa Leaves’ Extract (Eriobotrya japonica (Thunb) Lindl) from Tanah Karo.

DOI: 10.5220/0010102110761081

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

1076-1081

ISBN: 978-989-758-449-7

promote the use of natural and low-cost formulated

hand sanitizer.

2 MATERIALS AND METHODS

2.1 Plant Materials

Fresh Biwa (E. japonica) were collected from Suka

village, Tiga Panah district, Kabanjahe city, North

Sumatera. The parts used in this study were their

leaves. The sample was authenticated by Herbarium

Medanese, Universitas Sumatera Utara, Medan,

Indonesia.

2.2 Extraction Procedure

Fresh leaves were cleaned in flowing tap water,

shade-dried and pulverized into coarse powder. One

hundred grams (100 g) of powder were macerated

with ethanol, ethyl acetate and hexane (1:1) and

homogenized using shaker for 4 hr, settled down for

24 hr. Macerate were filtered using Whatman filter

paper and concentrated using rotary evaporator. The

crude extracts were subjected to antibacterial test in

Dimethyl Sulfoxide (DMSO) solution to obtain series

of concentration 5, 10, 15 and 20%.

2.3 Antibacterial Test

Series concentration of biwa extracts were tested

against S.aureus using disk diffusion method.

Overnight culture broth of S.aureus (Optical Density/

600

= 0.1 ≈ 10

CFU/mL) were swabbed on Muller

Hinton Agar (MHA) medium to obtain the bacteria

lawn. Sterile disks impregnated with 10 µL of

extracts were placed on top of medium with three

replications. The plates were incubated for 24 hr in

ambient condition. Halo zones around the disks

expressing the inhibitory effect to tested bacteria,

were measured using a caliper in millimeter unit

(mm).

2.4 Toxicity Test

Larva of Artemia salina were pre-hatched using

procedure from Muaja. (2013). Stock solutions of

extracts in DMSO with concentration of 20000, 2000,

200 and 20 ppm were pipetted into vials containing

20 live larva with final volume in each vials reaching

5 mL. Control vials were made by replacing extract

solution with saline solution. Vials were incubated for

24 hr in proper condition. Dead and live larva were

counted post-incubation. Data were analyzed using

standard probit analysis with 95% confidence interval

(Wahyuni., 2015).

2.5 Gel Formulation and Antibacterial

Test

The composition of three gel formulations are listed

in table 1. Antibacterial test was performed according

to previous procedure.

2.6 Antiseptic Test of Formulated and

Commercial Gel

Antiseptic test was conducted by using replica-

plating method [1]. Hands were washed in flowing

tap water and air-dried. Thumb were gently pressed

on top of Mannitol Salt Agar (MSA) to expose

indigenous S.aureus and serve as control plate.

Treatments (formulated gels 15 and 20%, blank

(negative control) and commercial gel (positive

control) were administered as the same procedure

stated before. Plates were incubated at 28

C for 24 hr.

After incubation, bacterial colonies were observed

and counted. Experiments were conducted in five

replications.

Table 1: Three gel formulations and their compositions.

Materials Formulati

on-1

Formulati

on-2

Formulati

on-3

Biwa

leaves’

extract

0% 15% 20%

Carbopol

940

0,5% 0,5% 0,5%

TEA 0,5% 0,5% 0,5%

Glycerine 1% 1% 1%

Sodium

metabisulp

hite

0,2% 0,2% 0,2%

Aquadest 200 mL 200 mL 200 mL

3 RESULTS AND DISCUSSION

3.1 Phytochemical Screening Results of

Biwa Extracts

The phytochemical compounds detected in Biwa

extracts are presented in Table 2. Flavonoid and

tannin were detected only in ethanolic extract

exposing that ethanol may be used in future extraction

Antibacterial-based Hand Sanitizer of Biwa Leaves’ Extract (Eriobotrya japonica (Thunb) Lindl) from Tanah Karo

1077

procedure to obtain desirable compound from

flavonoid and tannin groups.

Table 2: Phytochemical compound groups detected during

screening.

Solvents Flavonoi

Tannin

Ethanol + +

Ethyl Acetate - -

Hexane - -

Flavonoid is a group of phytochemical

compounds C15 consisting of two carbon cores with

three carbon units. Flavonoids are also known as

bioactive compound in plants that can be used as

antibacteria (Suteja., 2016). Tannins are polyphenol

compounds with a high molecular weight comprising

hydroxy and carboxyl groups. Tannins consisted of

two types: condensed and hydrolyzed tannins. Other

study reported that the tannin compound in the extract

of Averrhoa bilimbi L. was potential as antibacteria

against Staphylococcus aureus and Escherichia coli

bacteria Ummah (2010). Since the desirable

compounds were only detected in ethanolic extract,

ethyl acetate and hexane extracts were not subjected

to antibacterial test.

3.2 Antibacterial Activity of Biwa

Ethanolic Extracts against

Staphylococcus Aureus

The results of antibacterial activity of ethanolic

extracts are given in Table 3. Concisely, the use of

higher concentration of extracts increased the

diameter of halo zones in this study, explaining the

more toxicity of solutions against tested bacteria

(Figure 1).

Table 3: Antibacterial activity of different concentrations of

Biwa extracts against S. Aureus.

Diameter of

Inhibition Zone

(

)

Chloram

henicol 30.00

Alcohol 70% 7.00

DMSO 7.00

Ethanolic extract 5% 6.00

Ethanolic extract 10% 6.30

Ethanolic extract 15% 8.00

Ethanolic extract 20% 9.60

1 2

3 4

Figure 1

Representative images of inhibitory zones from

plate: A. Positive (chloramphenicol) and negative control

(alcohol 70% and DMSO), B. Concentration 5%, C.

Concentration 10%, D. Concentration 15% and E.

Concentration 20%.

Mechanism of antibacterial phenolic components

e.g. steroids, alkaloids, triterpenoids, flavonoids and

saponins, generally interact with intracellular protein

or cytoplasmic walls via hydrogen bonding and

hydrophobic interactions (Naidu and Davidson,

2000). Another mechanism is by interfering

enzymatic activity of cells through the diffusion of

polar extracts into cell interior.According to

Siswandono and Soekardjo (2000), the concentration

of certain compoundwith antibacterial properties is

determined from its inhibition to bacteria growth in

plate assay. In addition, the size of the inhibitory zone

may be influenced by several factors: test

microorganism (bacterial strain and physiological

condition), culture medium, method and diffusion

nature of the substances.

3.3 Result of Toxicity Test using Brine

Shrimp Lethality Test (BSLT)

The ethanolic extract were tested for its toxicity

against model organism to evaluate the safety issue as

A

B

c

U1

U2

U3

U1

U2 U3

U1

U2

U3

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

1078

active ingredient later for practical use as hand

sanitizer. The results are presented in Table 4. Similar

to previous result, the 10-fold increased concentration

of extracts led to increase of dead larva in this

experiment.

Table 4: Percentage of mortality during exposure to Biwa

extracts.

Concent

ration

(ppm)

Repl

ica 1

Repl

ica 2

Repl

ica 3

Mean

Mortal

ity

0 0 0 0 0 0%

20 3 3 5 3,66 37%

200 5 4 6 5 50%

2000 6 5 8 6,33 63%

20000 8 9 9 8,66 87%

The result of probit analysis showed that LC

value of Biwa leaves’ extract was 162,18 ppm.Meyer.

(1982) stated that an extract to have a potential toxic

activity in the BSLT toxicity if the extract caused the

death of 50% of the test animals at concentrations

<1000 ppm. It can be stated that leaf biwa extract is

toxic with LC

value obtained at concentration

162,18 ppm.

3.4 Characterization of Gel

Formulation

Antiseptic gel formulation showed good

characteristics to be used as topical hand sanitizer

with details in table 5.

Table 5: Characteristics of gel formulation.

From table 5, it can be seen that pH of formulated

and commercial gel is below physiological skin pH.

Skins are covered with acidic mantle which make the

surrounding pH between 4.5–6.5 (Tifaley., 2007).

The more acidic or basic condition will alter the skin

conditions from drying, sensitive, and easily

penetrated by microbes or infection.

3.5 Antibacterial Activity of Gel

Formulation

Biwa leaves’ extracts in gel formulation were tested

against Staphylococcus aureus.The gel showed

inhibitory activity yet showing the retain of

antibacterial properties from extracts. The results can

be seen in Table 6.

Table 6: Results of antibacterial test between formulated

and commercial gels.

Sampel

Replica

Replica 2

Mean

(mm)

Commercial gel

(Alcohol 70%)

6.60 6.60 6.60

Blank (K-) 0.00 0.00 0.00

Gel formulation

15%

6.20 6.50 6.30

Gel formulation

20%

7.00 6.60 7.00

From the results, it can be seen that antibacterial

activity between formulated and commercial gels

were not so different in terms of diameter of

inhibition zone against S.aureus. Antibacterial

activity produced from commercial or alcoholic gel

was not quite effective since it is volatile yet reducing

the effectiveness upon administration.

1 2

Figure 2 Representative images of inhibitory zones from

plate:K- (Blank), 15% (gel formulation), 20% (gel

formulation), K+ (Commercial gel); 1 and 2 (Replica 1 and

2).

Formul

ation

Characters

Flavor Colour Appear

ance

pH Visc

osit

Alkoho

l 70%

Citrus White Semiso

lid

5 173

7,02

Formul

a 1

(Blanko

)

None White Semiso

lid

5 238

28,8

Formul

a 2

(15%)

Extract Green Semiso

lid

6 448

7,75

Formul

a 3

(20%)

Extract Green Semiso

lid

7 203

5,67

‐

15%

20%

K+

‐

20%

15%

K+

Antibacterial-based Hand Sanitizer of Biwa Leaves’ Extract (Eriobotrya japonica (Thunb) Lindl) from Tanah Karo

1079

3.6 Antiseptic Test of Gel Formulation

The results of efficacy test using gel formulation as

antiseptic can be seen in Figure 3 and 4. The average

number of colonies observed from gel formulation is

17.4 colonies from concentration 20%. Although our

results showed considerable decrease of S.aureus

colonies between formulation and control, it still not

compensate with the commercial gel. In case of safety

use, we still promote biwa formulated gel to be used

as hand sanitizer for future application.

Figure 3: Result of efficacy test using gel formulation as

compared with blank, control and commercial antiseptic.

1 2

3 4

Figure 4 Antiseptic test of Biwa leaves’extract as

compared with blank, control and commercial gels in

Mannitol Salt Agar (MSA) with five replications.

4 CONCLUSIONS

Biwa leaves’ ethanolic extract contained flavonoid

and tannin groups in phytochemical screening with

potential antibacterial activity against S.aureus at

concentration of 15 and 20% with diameter of

inhibition zone measuring 8.00 and 9.60 mm. The

of extracts obtained in this study was 162.18

ppm showing less toxicity. Gel formulation with

concentration of 20% extracts decrease the

population of S.aureus as many as 17.4 colonies.

ACKNOWLEDGEMENTS

The authors would like to express the highest

gratitude to Universitas Sumatera Utara, Ministry of

Research and Higher Education for the financial aid

through scheme of Program Kreatifitas Mahasiswa–

Penelitian (PKM-P) year 2018. Special thank to

Laboratory of Microbiology, Inorganic Chemistry

and Pharmacy for their permission on resource

sharing during laboratory experiments.

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